Color discrimination in the red range with only one long ...phylogeny. The phylogeny is based upon a...

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IntroductionColor vision enables animals to reliably detect and recognize

important objects such as food sources, hosts or matingpartners (for reviews, see Menzel, 1979; Kelber et al., 2003).It arises from the comparison of signals from at least two typesof photoreceptor with different spectral sensitivities.Depending on the number and sensitivities of photoreceptortypes, the spectral range of color vision differs between speciesand is usually narrower than the total range of vision. Mostinsects have a short wavelength (S) receptor sensitive in theultraviolet (UV) range, a medium wavelength (M) receptorsensitive in the blue range and only one long wavelength (L)receptor sensitive in the green/yellow range. In the case of thehoneybee, the best studied example, these receptors have peaksensitivities that correspond to 344·nm (S), 436·nm (M) and544·nm (L) (Peitsch et al., 1992). Moths with a receptor setsimilar to that of the honeybee are incapable of discriminatingbetween lights of 590 and 630·nm by means of colors butinstead they used the intensity of the stimuli (Kelber andHénique, 1999).

The spectral sensitivity primarily depends on the visualpigment expressed in the receptor. The three insect receptor

types correspond to three major clades of the insect opsinphylogenetic tree (Fig.·1). In bees, moths and butterflies, eachommatidium has six (or seven) receptors expressing L opsin,and two receptors expressing either both M opsin, both S opsinor one M opsin and one S opsin: Vanessa cardui (Briscoe etal., 2003); Danaus plexippus (Sauman et al., 2005); Pierisrapae (Arikawa et al., 2005; Wakakuwa et al., 2004); Manducasexta (White et al., 2003); Bombus terrestris (Spaethe andBriscoe, 2005); Apis mellifera (Wakakuwa et al., 2005).Papilionid butterflies have evolved a fourth, red-sensitive opsin(Fig.·1) expressed in four receptors in a subset of ommatidia(Arikawa, 2003), and behavioral experiments have proved thatthis enables them to discriminate colors in the L range, e.g.spectral lights of 590 and 640·nm wavelength (Kelber andPfaff, 1999).

The presence of molecules other than opsins in the receptorcells can filter the light traveling inside the rhabdom, modifyingthe spectral sensitivity of the receptor (Goldsmith and Bernard,1974; Kong et al., 1980). In butterflies (with the exception ofpapilionids) a tapetum basal to the rhabdom reflects eitherbroadband light (300–700·nm) as in Vanessa cardui (Briscoe etal., 2003), or relatively narrow-band light (320–590·nm)

The basic precondition for color vision is the presence ofat least two receptor types with different spectralsensitivities. The sensitivity of a receptor is mostly definedby the opsin-based visual pigment expressed in it. Weshow here, through behavioral experiments, that thenymphalid butterfly Heliconius erato, although itexpresses short and medium wavelength opsins and onlyone long wavelength opsin, discriminates colors in thelong-wavelength range (590·nm, 620·nm and 640·nm),whereas another nymphalid, Vanessa atalanta, despitehaving color vision, is unable to do so. In the eyes of H.erato we identified filtering pigments very close to therhabdom which differ between ommatidia and producethe yellow and red ommatidial reflection seen under

orthodromic illumination. The eyes of V. atalanta lack thefiltering pigments, and reflect a homogeneous orange. Wehypothesize that the filtering pigments found in the eyes ofH. erato may shift the spectral sensitivity peak of the longwavelength receptors in some ommatidia towards longerwavelengths. The comparison of the signals between thetwo new receptor types makes color discrimination in thered range possible. To our knowledge, this is the firstbehavioral proof of color vision based on receptorsexpressing the same opsin.

Key words: color vision, opsin, filter pigment, insect, butterfly,Heliconius erato.

Summary

The Journal of Experimental Biology 209, 1944-1955Published by The Company of Biologists 2006doi:10.1242/jeb.02207

Color discrimination in the red range with only one long-wavelength sensitiveopsin

Guillermo Zaccardi1,*, Almut Kelber1, Marilou P. Sison-Mangus2 and Adriana D. Briscoe21Vision Group, Department of Cell and Organism Biology, Lund University, Helgonavägen 3, S-22362 Lund, Sweden

and 2Comparative and Evolutionary Physiology Group, Department of Ecology and Evolutionary Biology,University of California, Irvine, CA 92697, USA

*Author for correspondence (e-mail: [email protected])

Accepted 13 March 2006

THE JOURNAL OF EXPERIMENTAL BIOLOGY

1945One opsin two receptors

(Arikawa et al., 2005; Douglas and Marshall, 1999; Stavenga,2002a) (G. Bernard, personal communication). In the latter case,it can thus modify the spectral sensitivity of the photoreceptors.However, the effect of a tapetum is limited since it only affectsthe spectral composition of light during the second pass throughthe retina, and most light absorption happens on the first pass,which is before light reaches the tapetum.

Different filtering pigments associated with differentphotoreceptors that express the same opsin can result inphotoreceptors with different spectral sensitivities that can beused for color vision. Additional photoreceptors resulting fromthis kind of filtering would not extend the total spectralsensitivity of the animal – that is defined by the sensitivities ofthe opsin pigments – but it may extend the range of color vision

Fig.·1. Arthropod opsin genephylogeny. The phylogeny is basedupon a neighbor-joining analysis offirst and second nucleotide positionsusing the Tamura–Nei model ofevolution with a correction forheterogeneous patterns of evolutionamong lineages. A total of 631nucleotide sites were used. Numbersat the junctions indicate bootstrapreplicates out of 1000 (given as apercentage) in which a particular nodeis supported. Red indicates clonedHeliconius erato opsin cDNAs.GenBank accession numbers for thesequences used in the reconstructionare as follows. Chelicerates: Limuluspolyphemus (lateral eye, L03781;ocelli, L03782). Crustaceans:Procambarus clarkii (S53494).Insects: Anopheles gambiae [accessionno. is given as supplementaryinformation in Hill et al. (Hill et al.,2002)]; Antheraea pernyi (AB073299);Apis mellifera (UV, AF004169;Blue, AF004168; LW, U26026);Bombyx mori (LWRh1, AB064496),Camponotus abdominalis (LW,U32502; SW, AF042788); Cataglyphisbombycinus (LW, U32501; SW,AF042787); Danaus plexippus (UVRh,AY605546; BlueRh, AY605545;LWRh, AY605544); Drosophilamelanogaster (Rh1, K02315; Rh2,M12896; Rh3, M17718; Rh4, M17730;Rh5, U67905; Rh6, Z86118);Homalodisca coagulata (AY588065);Manduca sexta (Manop1, L78080;Manop2, L78081; Manop3,AD001674); Megoura viciae (UV,AF189715; LW AF189714); Papilioglaucus (PglRh1, AF077189; PglRh2,AF077190; PglRh3, AF067080;PglRh5, AF077191; PglRh6,AF077192); Oncometopia nigricans(AY725781); Pieris rapae (PrL,AB177984); Schistocerca gregaria(Lo1, X80071; Lo2, X80072);Sphodromantis spp. (X71665). Full-length nucleotide sequences for the Bombyx mori UV, blue and L opsin coding regions were obtained using a tBlastx search of GenBank wholegenome sequences (wgs), manually removing the introns in MacClade, and then comparing the coding sequences with partial B. mori opsincDNAs reported in (Shimizu et al., 1998). The bar indicates the number of substitutions/site.

Bombyx mori UVRh Manduca sexta Manop2

Heliconius erato UVRh Danaus plexippus UVRh

Papilio glaucus PglRh5 Bombus impatiens UVRh Apis mellifera UV Camponotus abdominalis LW Anopheles gambiae Drosophila melanogaster Rh3

Drosophila melanogaster Rh4

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Manduca sexta Blue Bombyx mori BlueRh

Papilio glaucus PglRh6 Heliconius erato BlueRh

Danaus plexippus BlueRh Schistocerca gregaria Lo2

Apis mellifera Blue Drosophila melanogaster Rh5

Blue

Drosophila melanogaster Rh2 Drosophila melanogaster Rh1 Limulus polyphemus ocelli Limulus polyphemus lateral eye

Procambarus clarkii Apis mellifera LWRh2

Drosophila melanogaster Rh6 Oncometopia nigricans Homalodisca coagulata

Megoura viciae LW Sphodromantis spp. Schistocerca gregaria Cataglyphis bombycina Camponotus abdominalis

Apis mellifera LWRh1 Danaus plexippus LWRh

Heliconius erato LWRh Pieris rapae PrL

Papilio glaucus PglRh2 Papilio glaucus PglRh3

Papilio glaucus PglRh1 Bombyx mori LWRh1

Manduca sexta Bombyx mori LWRh2

Antheraea pernyi

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(i.e. the range of wavelengths that can be discriminated). Thecolored oil droplets found in some birds had earlier beenthought to play this role in color vision (Walls, 1942; King-Smith, 1969) but it is now known that each colored oil dropletis associated with a specific opsin in the receptor. They act asa cut-off filter, narrowing the spectral sensitivity of the cones,rather than increasing spectral types (Vorobyev, 2003). Thesame principle applies to the lateral filtering pigments in thereceptors of Papilio xuthus (Arikawa et al., 1999). However,in the eyes of the male small white butterfly, Pieris rapae, thepale-red or deep-red pigment clusters that surround therhabdoms of different ommatidia act as long-pass filters,creating receptors with peak sensitivity at 620 or 640·nm, butboth contain the same opsin (Qiu et al., 2002; Qiu andArikawa, 2003; Wakakuwa et al., 2004). In these cases, colordiscrimination could theoretically be extended by means offiltering pigments but direct behavioral evidence confirmingthis is missing. Different corneal filters in different ommatidiafound in tabanid flies and grasshoppers have the same potentialto create new receptor types (Kong et al., 1980; Lunau andKnüttel, 1995).

The ability to discriminate colors in the red range seems tobe very useful. It can increase the number of flower speciesthat can be distinguished and facilitate the finding of betterhosts for larvae. This seems to be the case in Papilio butterflies.These animals use color vision not only when foraging forflowers (Kelber and Pfaff, 1999; Kinoshita et al., 1999) but alsowhen making decisions about where to oviposit (Kelber, 1999).Color discrimination in the L range enables them to choose theoptimal host for their offspring. The same probably applies toPieris butterflies (Scherer and Kolb, 1987; Kelber, 2001; Weissand Papaj, 2003).

All species investigated so far within the third butterflyfamily, Nymphalidae, have only three opsin genes (Sauman etal., 2005; Briscoe and Bernard, 2005) but more than threereceptor types have been reported from several species(Bernard, 1979; Kinoshita et al., 1997; Stavenga et al., 2001)(for a review, see Briscoe and Chittka, 2001). The sensitivitycurves recorded from the nymphalid butterflies Polygonia c-aureum and Sasakia charonda show clearly that the long-wavelength cut-off of all L receptors coincide, whereas peaksensitivities differ by as much as 50·nm. This can only resultfrom filtering, not from multiple opsins (Kinoshita et al.,1997).

We have chosen to investigate whether color vision extendsinto the L range, in two species of the nymphalid family,Vanessa atalanta (Linnaeus 1758; subfamily Nymphalinae)and Heliconius erato (Linnaeus 1758; subfamilyHeliconiinae). Both species have red areas on their wings, thuscolor discrimination in the red range could be useful for matedetection as well. In H. erato only three receptor types havebeen described so far, with �max at 370, 470 and 570·nm(Fig.·2) (Struwe, 1972). However, electroretinograms(Bernhard et al., 1970; Swihart and Gordon, 1971) andelectrophysiological recordings in the brain (Swihart, 1972)give indications that a second L receptor (�max at 620·nm) may

G. Zaccardi and others

exist. It has long been known that Heliconius uses color to findfood (Swihart, 1971), and both color and polarized light cuesare used by males in choosing mates (Jiggins et al., 2001;Sweeney et al., 2003). V. atalanta has three receptors with �maxat 360, 470 and 530·nm similar to the congener V. cardui(Fig.·2) (Briscoe et al., 2003) (G. Bernard, personalcommunication). We have performed behavioral experiments,characterized the opsin genes and their expression in the retina,and studied the eye glow and lateral filtering pigments. Inshort, we prove, for the first time using behavioral studies, thatan insect with only S, M and L opsin pigments can discriminatecolors in the red range. This is not explained by the sensitivitiesof the opsins alone and it is probably due to the shift in thereceptors’ sensitivity caused by the presence of lateral filteringpigments.

Materials and methodsAnimals

Heliconius erato butterflies were bought as pupae from aprofessional breeder (Stratford Butterfly Farm, England). Thepupae were hung on pieces of cardboard in plastic boxes andkept at high humidity at 28–30°C under a 12·h:12·h light:darkregime. Vanessa atalanta butterflies were reared in the labfrom animals captured locally around Lund (Sweden), and

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ityFig.·2. Spectral sensitivity of the photoreceptors calculated from thesensitivity maxima given in Struwe (Struwe, 1972) for H. erato (solidlines) and Briscoe et al. (Briscoe et al., 2003) for V. cardui (brokenlines) using the template from Stavenga et al. (Stavenga et al., 1993).Peak sensitivity values for the short, medium and long wavelengthvisual pigments are identical between V. cardui and V. atalanta (notshown; G. Bernard, personal communication). Observe that bothspecies have the medium wavelength photoreceptor peaking at470·nm therefore the curves for both species coincide. The verticaldotted lines correspond to the wavelengths of the stimuli used.



allowed to oviposit on nettle leaves (Urtica dioica), itsnatural host, which was also collected around Lund. Thecaterpillars were kept in plastic boxes and the nettle leaveswere changed every day. When V. atalanta butterfliespupated they were hung on pieces of cardboard in the samemanner as H. erato. The pupae were kept under a 20·h:4·hlight:dark regime at 25–28°C and high humidity. Once theadults of both species emerged, they were individuallymarked on the wings and released in a flight cage. Thebutterflies were fed with 20% (w/w) sucrose solution on thepositive stimulus during the training and test. On average,only about half the animals would eclose from the pupa. Ofthese animals, not all survived long enough to finish theexperiment. We started with 15 H. erato in one experiment,but we could only collect sufficient data with nine of them.In a second experiment, where we started training sevenanimals, four died before the end of the experiment. With V.atalanta, only three out of nine animals survived until theexperiment was finished.

Behavioral experiments

The experiments with H. erato were performed in an indoorcage (2·m�1.60·m�2.8·m) constructed from metal pipes andcovered with gauze except for the ceiling that was made of athin plastic sheet. The cage was illuminated by 26 fluorescenttubes (Osram Biolux 965, 65·W; München, Germany)distributed around and above the cage. The intensity of theillumination was approximately 100·cd·m–2 in the center of the

cage. The temperature was 30°C, and the light regime was setat 12·h:12·h light:dark. The experiments with V. atalanta wereperformed in a smaller cage (75·cm�50·cm�60·cm)illuminated by four fluorescent tubes (Philips TLD 965 18·W;Eindhoven, The Netherlands). The light intensity in the centerof the cage was roughly the same as in the big cage. The lightregime was 20·h:4·h light:dark, and the temperature between20 and 25°C.

The stimuli were presented vertically in an apparatus(Fig.·3A,B) consisting of a black metal plate measuring20·cm�10·cm. Two light guides were attached to the plateconnected to two independent cold light sources (Schott KL2500, Mainx, Germany). Each light beam passed through aninterference filter and a transparent Plexiglass feeder disk(Fig.·3B,C). These feeders were built such that the sucrosesolution could be placed away from the center and the animalswere not able to see the drop of sugar solution (Fig.·3C). Frontallytwo colored disks of 3·cm in diameter separated by 6·cm werevisible. We used four different colors as stimuli. The colors wereobtained by means of four narrow band (10·nm bandwidth)interference filters, transmitting light of 440·nm, 590·nm, 620·nmand 640·nm. Fig.·2 shows these wavelengths together with thesensitivity curves of each receptor in both species. The lightintensities could be adjusted electrically, by changing the energydelivered to the light bulb, and mechanically, by means of adiaphragm. Using these two possibilities together, a large rangeof intensities (between 3.19�1015 and 2.75�1017;quanta·s–1·steradian–1·cm–2) could be attained.

Training and test

The butterflies were fed for the first timebetween 6·h and 8·h after they emerged. Adrop of sugar water was placed on the feederilluminated with the positive color (+); theother feeder (–) was always kept empty. Eachbutterfly was grasped by the wings and theproboscis was unrolled with a thin needleuntil it touched the sugar solution.Immediately the butterfly started to drink.This procedure was repeated twice a day.After 3–4·days, the animals flew towards theapparatus by themselves and were able to findthe rewarded color (+). From this moment, thechoices were registered. Each time an animalapproached and touched a feeder, either withits proboscis or with a tarsus, a choice wasregistered. If an animal touched the feedermore than once during an approach, only thefirst touch was counted. In further dataanalysis, we only included animals that madea minimum number of 15 choices with eachintensity combination, thus in total at least 60choices.

We first trained both species to discriminatea yellow or red (590·nm or 620·nm) and a blue(440·nm) color, to determine whether the

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Fig.·3. Schematic views of the apparatus used to train and test the butterflies to thedifferent colors. (A) Frontal view of the apparatus. (B) Lateral view of the apparatus.(C) The feeder disk.


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animals used color vision at all. The animals that were able todiscriminate these colors were then trained to a second pair ofwavelengths. Animals did not survive long enough to be trainedto three wavelength combinations. Therefore, H. erato that weretrained to two combinations of long wavelengths were notinitially trained with blue and yellow. These H. erato were firsttrained to 620·nm (+) vs 590·nm. When they reached theminimum number of choices, the same individuals were trainedto 620·nm (+) vs 640·nm. V. atalanta were first trained to620·nm (+) vs 440·nm, and once the animals reached theminimum number of choices, the same individuals were trainedto 620·nm (+) vs 590·nm. Only one animal at a time wasallowed to visit the apparatus. Each butterfly was allowed todrink for 1–2·s and then gently forced to leave for a new choice.Between choices the intensities and/or the position of thestimuli were changed in a pseudorandom way. Five differentratios of the physical intensities of +/– were used: 0.01, 0.1, 1,10 and 100 (i.e. +100 times less intense than –, +10 times lessintense than –, equal intensities and +10 times more intense thanthe –, +100 times more intense than –, respectively), but onlythree or four of them were used with one wavelengthcombination. This was achieved by changing the intensities ofboth the rewarded and the unrewarded stimulus. This schemaresulted in an average of 10 choices per animal daily.

The performance of each animal with each intensitycombination was evaluated separately by comparing thenumbers of choices this animal made with the critical valuecorresponding to two-tailed significance levels (�) of 0.05 fora binomial proportion of P=0.5 [equal number of choices foreach color; Rohlf and Sokal (Rohlf and Sokal, 1995), p. 107,Table·Q].

Light microscopy

Under daylight illumination the heads of 10-day old H. eratowere severed in two halves and the pieces were put in 4% PFA(paraformaldehyde) in 0.1·mol·l–1 phosphate-buffered saline(pH·7.2) for 1·h. The eyes were dehydrated in an alcohol series(50%, 75%, 96%, 100%), finally immersed in 100% acetoneand embedded in Epon resin (Agar Scientific, Agar ScientificLtd, Essex, UK) After hardening the resin at 60°C for 48·h theeyes were cut laterally in 10·�m thick sections using amicrotome and mounted on a slide. In this way the ommatidiathat in the living animal pointed sideways, pointed directly atthe observer in the microscope. Some H. erato were darkadapted by keeping them in total darkness for 30·min at roomtemperature and then for 20·min in total darkness at 4°C. Afterthat, the head of the animal was cut off under red light(produced by attaching a 660·nm cut-off filter to the tip of alight guide connected to a Schott KL 2500 lamp) in order toavoid light adapting the eyes. V. atalanta butterflies werecaptured locally around Lund (Sweden) and were treated in thesame way as the light adapted H. erato.

Eye glow

At least four dark-adapted H. erato and V. atalanta eyeswere photographed with a Nikon camera connected to a


modified epi-illumination microscope. In this instrument, theincident light applied to the eye is channeled by the facet lensand crystalline cone into the light-guiding rhabdoms (see Land,1984; Miller and Bernard, 1968; Stavenga, 2002b). When thedark-adapted eye is illuminated with strong light and observedfrom the same direction (orthodromic illumination), a brighteye-glow is seen for a few seconds. Light reaching theommatidial tapetum is reflected and guided back through therhabdom. When not absorbed there, it leaves the eye again andis then observable as the eye glow. The butterflies wererestrained by waxing the thorax and head to a support but wereotherwise alive and intact. The animals were oriented such thatpictures could be taken in the frontal-lateral part of the eye.The eye glow was photographed by leaving the shutter of thecamera open and delivering flashes of between 0.1·s and 0.5·swith intervals of 10·s. In this way, the eye glow could bephotographed without the pupil closing. As a light source weused a xenon arc lamp that supplied 1.4�10–4·W·cm–2. Theobjective lens was a Zeiss Luminar 25·mm (3.5/A·0.15). Tostudy the spectral sensitivity of the pupil response, weilluminated the eye with lights of different wavelengthsobtained by means of interference filters (680·nm, 650·nm,620·nm, 590·nm; 10·nm bandwidth) and observed pupilclosure.

PCR, cloning and sequencing

The H. erato opsins were isolated by the polymerase chainreaction (PCR) using a combination of degenerate and gene-specific primers. cDNA template was prepared from RNAextracted from whole head tissue (Trizol; Gibco-BRL,Gaithersburg, MD, USA) and synthesized using the MarathoncDNA Amplification Kit (BD Biosciences Clontech, MountainView, CA, USA). PCR products were ligated into the pGEM-T Easy cloning vector (Promega, Madison, WI, USA) andplasmids were screened by EcoRI digestion for inserts of thecorrect size. Plasmid DNA was cycle sequenced using the BigDye Terminator 3.1 Cycle Sequencing Kit (AppliedBiosystems, Foster City, CA, USA) and the core sequencingfacilities at the University of California, Irvine. In this initialscreen, we sequenced more than 100 clones, and then designedopsin gene-specific primers to use in a multiplex PCR. Toensure that all opsins had indeed been recovered,approximately 120 additional plasmids were screened usingmultiplex PCR in which three pairs of opsin gene-specificprimers were combined in a single PCR reaction. Only thoseplasmids that did not amplify with those primers were thensequenced.

Phylogenetic analysis

To identify the homology of the cloned H. erato opsins, weconducted a BLAST search and downloaded a total of 41 full-length arthropod opsin genes from GenBank includingrepresentatives of all available insect orders (six total), as wellas chelicerates and crustaceans. Only first and secondnucleotide positions were used as third positions were saturated(A.D.B.,, unpublished observation). A gene tree was



reconstructed using the neighbor-joining algorithm withTamura-Nei distance, heterogeneous rates among lineages andcomplete deletion of gaps as implemented in MEGA·3.0(Kumar et al., 2004). Robustness of the tree was assessed bybootstrap analysis (1000 replicates).

Cryosectioning

The adult butterflies were placed at 4°C for 1·h before beingkilled by a swift severing of the head with a scalpel.Subsequently, the head was cut in half. The eyes were fixed in4% PFA in 1� phosphate-buffered saline (pH·7.2) for 2·h to4·h at room temperature and stepped through a sucrose gradient(10%, 20%, 30%). Then the tissue was cryostat sectioned into8–12·�m slices at –18°C and placed on a slide. The eyes wereoriented such that the ommatidia that in the living animalpointed directly to the front, pointed directly at the observer.

Riboprobe synthesis and in situ hybridization

Starting with 1·�g of purified PCR product (amplified fromplasmid DNA), digoxigenin-UTP-labeled RNA probes(riboprobes) complementary to the mRNAs of the visualpigments were synthesized by using a DIG RNA labeling kit(Roche Diagnostics, Mannheim, Germany). After thesynthesis, the riboprobes were precipitated with 4·mol·l–1 LiCland 100% ethanol. A dot blot procedure was used to quantifythe amount of riboprobe. Typically, 10·ng/�l of riboprobe wasobtained after this procedure.

The slides with the sections were then incubated inhybridization buffer (0.3·mmol·l–1 NaCl, 2.5·mmol·l–1 EDTA,20·mmol·l–1 Tris-HCl, pH·8.0, 50% formamide, 10% dextransulfate, 100·g·ml–1 yeast tRNA and 1� Denhart’s medium)(Sakamoto et al., 1996) in a humid chamber for 30·min at60°C. The labeled probe was diluted in the hybridization buffer(1:75), corresponding to approximately 0.013·ng of probe perml of hybridization buffer. The sections were incubated in thediluted probe overnight at 55–60°C in a humid chamber andthen washed with 2�, 1� and 0.1� standard saline citrate and0.1% Tween 20, for 10·min each. The probes were identifiedin the histologic sections by incubation with an anti-digoxigenin alkaline phosphatase-conjugated antibody(Boehringer Mannheim), diluted in 1� phosphate buffer plusTween 20 (1:1000) for 2·h. The probes were detected by acolorimetric reaction produced by Nitro Blue tetrazolium (5-bromo-4-chloro-3-indolylphosphate) and 10% Tween 20 inalkaline phosphatase developing solution. An Axioskopmicroscope (Zeiss, Thornwood, NY, USA) equipped with anAxioCam Hrc digital camera (Zeiss) was used to collectimages. Image data were recorded in ZeissVision 3.1 softwareon a personal computer at 2,060�2,600 pixel resolution.

ResultsBehavior

In order to investigate whether H. erato or V. atalanta havecolor vision in the L range, we first determined that the animalswere capable of discriminating a rewarded color from an

unrewarded color. Fig.·4A shows that V. atalanta (N=3) wasable to discriminate a rewarded light stimulus of 620·nm (+)from 440·nm. The animals chose it significantly more oftenthan the unrewarded one independently of the intensity (threeanimals, at least 15 choices in each intensity combination,P

1950 G. Zaccardi and others

Different kind of opsins and their expression in the retina

In order to see if the capability of H. erato to discriminatebetween stimuli of long wavelengths is based on the expressionof more than one L opsin we screened a cDNA librarysynthesized from adult eyes. More than 200 clones werescreened and only one L opsin-encoding mRNA was found inthe compound eyes of H. erato, along with blue and ultravioletopsin-encoding mRNA transcripts. The GenBank accession

numbers for these genes are as follows: UVRh, AY918904;BlueRh, AY918906; LWRh, AY918907. To see how thesethree opsins are expressed, at least 50 eyes were analyzed afterperforming in situ hybridization in the frontal and fronto-lateralpart of the compound eye. Fig.·5 shows an example of thehybridization pattern obtained after using UVRh, BlueRh andLWRh digoxigenin-labeled antisense riboprobes. Fig.·5Ashows the pattern produced by the UVRh riboprobe. Some

Fig.·4. Choice frequencies of H. erato and V. atalanta for the four colors after training, as a function of the ratio between the intensities of therewarded color and the unrewarded color. The symbols represent the individual performance and the line the average. (A) Three V. atalantatrained to 620·nm as the rewarded color and 440·nm as unrewarded. All choices differ significantly from chance (P


ommatidia show two cells stained, others show only one cellstained and some no staining. The same configuration can beobserved when the BlueRh riboprobe was used (Fig.·5B). Sincethe sections in Fig.·5A,B are consecutive, the position of thecells in both pictures can be followed (black circles in the insetsmark the same ommatidia). We observed three different typesof ommatidia. The ommatidia that express UVRh in two cellsdo not express BlueRh. Reciprocally, the ommatidia thatexpress BlueRh in two cells do not express UVRh. The thirdtype of ommatidia expresses each opsin mRNA in one cell.This is the same situation as in V. cardui (Briscoe et al., 2003),P. xuthus (Arikawa, 2003), Pieris rapae (Arikawa et al., 2005;Wakakuwa et al., 2004), Manduca sexta (White et al., 2003),and in Danaus plexippus (Sauman et al., 2005). Fig·5C,D showthe LWRh pattern, in which six cells are stained in allommatidia across the whole section. Each cell expresses onlyone of the three opsins, and we found no evidence of co-expression. This pattern was seen in all the eyes examined. We,therefore, conclude that the difference between H. erato and

V. atalanta in their ability to discriminate light in the longwavelength part of the spectrum cannot be attributed to thepresence of a second L opsin.

Eye glow and histology: Lateral filtering pigmentheterogeneity in H. erato

Since H. erato can extend their color vision range into thelong wavelengths using only one L opsin which ishomogeneously distributed, we next studied whether theommatidia differ in aspects other than the S and M opsinexpression. We first examined the light that is reflected by thetapetum and emerges from the compound eye (eye glow) of H.erato, using an ophthalmoscope, and found two classes ofommatidia (Fig.·6A). One class of ommatidia reflects yellowlight and the other reflects red. By contrast, all ommatidia ofV. atalanta reflect an homogeneous orange (Fig.·6D) in thesame fashion as in V. cardui, a close relative (Briscoe andBernard, 2005).

When we applied lights of different wavelengths by placing

Fig.·5. In situ hybridizations ofcryostat sections of the compoundeye of H. erato using UVRh,BlueRh and LWRh digoxigenin-labeled antisense riboprobes. (A)UVRh and (B) BlueRh opsinmRNAs, respectively, detectedusing an alkaline phosphatase-conjugated anti-digoxigeninantibody. The insets show themagnification of the boxed region.Since the sections are consecutive,the position of the sameommatidium in A can be identifiedin B, as indicated by aligning theblack circles. The butterfliesexpress these two opsin mRNAs inthree different ways in differentommatidia: S-S, S-M and M-M inthe R1 and R2 photoreceptor cells.(C) Expression pattern of theLWRh opsin mRNA transcript. Inevery ommatidium in the mainretina, six photoreceptor cellsexpress the L opsin mRNA. (D)Close up view of the L opsinmRNA expression pattern in theR3-8 photoreceptor cells. Sectionsare at around 160·�m from thecornea. Scale bars, 50·�m (A–C);20·�m (D).


1952

interference filters (680·nm, 650·nm, 620·nm,590·nm) between the light source and the eye of H.erato, we observed that the red and the yellowommatidia closed to different degrees (data notshown). Since the pupil closure is under the controlof the photoreceptors in each ommatidium(Stavenga, 1979), the different degrees of closure area result of different sensitivities of the receptors inboth ommatidial classes to these wavelengths.Considering the wavelengths of the test lights, weconclude that the L receptors must differ in theirspectral sensitivity between ommatidial classes, andthis could provide the basis for color vision in the Lrange. In V. atalanta all ommatidia close completelyand at the same speed when illuminated with 620·nmand 590·nm. When illuminated with 650·nm a partialclosure was observed indicating that thephotoreceptors are slightly sensitive to thiswavelength.

We hypothesized that, as has been shown in Pierisrapae (Wakakuwa et al., 2004) the occurrence of thetwo physiologically distinct H. erato ommatidialclasses might result from different populations oflateral filter pigments. Because of the waveguideproperties of the narrow butterfly rhabdom (Nilssonet al., 1988), lateral filter pigments can affect thewavelengths of light to which the receptors aresensitive (Miller and Bernard, 1968; Ribi, 1979;Stavenga, 2002a; Stavenga, 2002b).

We tested this hypothesis by inspecting serialsections through the compound eye, and found thatthe difference between the heterogeneous eye glowof H. erato and the homogeneous eye glow of V.atalanta may be due to differences in the presenceor absence, and distribution of lateral filter pigments.In a tangential section of the fronto-lateral region ofthe compound eye of H. erato (190·�m from thecornea), the presence of pupillary pigments can beobserved (Fig.·6B). In dark-adapted eyes thesepigments are further away from the rhabdoms thanin light adapted eyes (picture not shown). Thesepigments disappear completely around 220·�m andafter a gap, a second type of red pigmentationappears around 320·�m from the cornea. Thepigments at this depth (Fig.·6C, 370·�m from thecornea) are closer to the rhabdom than the pupillarypigments (distance between opposite pigment spots0.68±0.20·�m, mean ± s.d.; N=10) and do not moveas a function of light adaptation. Their coating of therhabdom means that they can filter the shortwavelength light traveling in the waveguide, andtherefore change the spectral sensitivity. The colorof this pigment is heterogeneous among differentommatidia (see circles marked a and b, in Fig.·6C). Thispigment heterogeneity is also evident at sections taken moreproximal in the eye (480·�m, picture not shown). We propose


that this heterogeneity of pigmentation may be the cause of theheterogeneous eye glow and the difference in speed of thepupil closure (i.e. different sensitivities).

Fig.·6. Eye glow, pupillary pigments and lateral filtering pigments of H. erato(A–C) and V. atalanta (D–F) from the fronto-lateral eye region. (A) Eye glowof H. erato. Two classes of ommatidia can be observed, one of them reflectsyellow and the other red. (B) Pupillary pigments at 190·�m from the cornea.(C) Lateral filtering pigments at 370·�m from the cornea vary betweenommatidia (circles a and b) compared to the uniformly distributed pupillarypigments. (D) Eye glow of V. atalanta. Only one orange reflecting class exists.(E) Pupillary pigments at 180·�m from the cornea. (F). Pupillary pigments at250·�m from the cornea. Uniform pupillary pigment density between ommatidiacan be observed in all photoreceptor cells. Sections are 10·�m thick. Scale bars,50·�m.



In the eye of V. atalanta (Fig.·6E,F), the only pigmentsdetected were the pupillary pigments (see also Stavenga,1979). We found no histological evidence of a lateral filteringpigment or pigment heterogeneity between ommatidia, whichis consistent with the homogeneous eye glow, the pupilclosure, experimental reflectance spectra (G. Bernard, personalcommunication), and with the data from the other species ofthis genus that was recently examined, V. cardui (Briscoe etal., 2003).

DiscussionColor discrimination in the long-wavelength range with one

opsin

We conclude that H. erato has color vision in the red range,based on two photoreceptor types containing the same opsin.The difference in the spectral sensitivities necessary toaccomplish color vision must result from other mechanismsthan a new opsin, and is probably the result of the expressionof different lateral filtering pigments in different ommatidia.

We have compared the color vision abilities of two butterflyspecies that possess three opsin genes coding for opsins in theS, M and L range. One of the species, V. atalanta, was able todiscriminate between blue (440·nm) and yellow (590·nm) withhigh accuracy but not between yellow (590·nm) and red(620·nm; Fig.·4A,B). This indicates that only one receptor typeis sensitive to both long wavelengths. The blue-sensitivereceptor is insensitive to these long wavelengths making acomparison of different receptor signals (and thus color vision)impossible. In the absence of a color difference, V. atalantaused the intensity of the stimulus as a choice criterion. Thisresult is similar to that obtained with a sphingid that alsopossesses three spectral types of receptors leaving it unable todiscriminate yellow from red (Kelber and Hénique, 1999).

In contrast, H. erato is able to discriminate 620·nm not onlyfrom 590·nm but also from 640·nm (Fig.·4C,D). Two spectraltypes of receptors must therefore be sensitive to light of620·nm wavelength. In P. aegeus, the ability to discriminate590·nm from 630·nm is mediated by a separate red receptorcontaining a separate red opsin (Kelber and Pfaff, 1999; Maticet al., 1983). This second L opsin evolved as a result of a geneduplication event, which occurred after the divergence of thenymphalid and papilionid lineages (Briscoe, 2001). In H.erato, our extensive search for an additional L opsin gene wasunsuccessful. Six of eight proximal photoreceptors in eachommatidium, and probably the ninth basal receptor express thesame known green-sensitive opsin (Fig.·5C,D). The remainingtwo receptors are two UV receptors, two blue receptors or oneof each (Fig.·5A,B). This situation is the same in V. cardui, aclose relative of V. atalanta (Briscoe et al., 2003). The bluereceptors of both species have a peak sensitivity at longerwavelengths than other insect blue receptors (470·nm) (Struwe,1972) [for comparison see Briscoe and Chittka (Briscoe andChittka, 2001)] but this cannot explain our results (Fig.·2). Weconclude that H. erato has color vision in the red range, withouthaving two opsins sensitive in this spectral range.

Do lateral filtering pigments create a second long-wavelengthreceptor?

The ophthalmoscope studies of H. erato revealed two classesof ommatidia differing in eye glow color (Fig.·6A). This issimilar to many butterfly eyes studied by Stavenga (Stavenga,2002a; Stavenga, 2002b), including another species of the largegenus Heliconius, H. melpomene. By contrast, a uniform eyeglow was seen in V. atalanta (Fig.·6D), similar to that of V.cardui and other nymphalids including Nymphalis antiopa,Siproeta steneles, Inachis io and Polygonia c-album (Briscoeand Bernard, 2005; Stavenga, 2002a). Possible mechanismsunderlying the different eye glow colors include lateral filteringpigments, different opsin densities and tapetal reflection.Different opsin densities are hard to prove; nonetheless, wecannot exclude them as a possible cause for the difference inthe ommatidial reflection seen in H. erato. Differences in tapetalreflection have little relevance since they only affect lightabsorbed on the way out of the receptor. This is a very smallamount as most light is absorbed on the way into the eye. Themost likely candidate causing differences between receptorspectra are the lateral filtering pigments found in H. erato butnot in V. atalanta (Fig.·6). These pigments are close enough tothe rhabdom to act as lateral filters. Different pigments indifferent ommatidia (Fig.·6C) can result in two kinds ofommatidia with different spectral sensitivities as has beenshown for P. rapae (Wakakuwa et al., 2004). Further studiesincluding electrophysiological measurements are needed toreveal the exact receptor sensitivities.

Color vision with filter pigments?

To our knowledge, H. erato is the first animal shown to usetwo photoreceptors containing the same opsin for color vision.We hypothesize that lateral filtering pigments may be the basisof the difference between the two receptor types in Heliconiusas well. Filtering pigments can extend the range of color visionby shifting the L receptor sensitivity in one class of ommatidia.The comparison between different receptors created in this wayallows the animal to discriminate colors at longer wavelengthsthan those expected by the opsins alone. The total range ofvision, which is set by the sensitivity of the opsins, is unaltered.The fact that Pieris and Papilio also have lateral filteringpigments (Arikawa, 2003; Wakakuwa et al., 2004) makes itprobable that this mechanism is older than the evolution of aseparate red opsin in Papilio, and common in butterflies. Thereis a high probability that butterflies such as Sasakia charonda,Polygonium c-aureum (Kinoshita et al., 1997) and Pieris rapaeare also able to discriminate colors in the red range. Given thereceptor curves measured (Wakakuwa et al., 2004), the colordiscrimination ability of P. rapae might extend even furtherinto the red range than color vision of H. erato.

Red filtering pigments that may change receptor sensitivityhave earlier been found in a hymenopteran insect (Ribi, 1978).We expect that careful studies will reveal more such cases andconclude that the study of opsin genes and their expression isnot sufficient to understand the evolution of color visionsystems in animals.


1954 G. Zaccardi and others

Special thanks to Prof Dan-Eric Nilsson for use of theophthalmoscope, Carina Rasmussen for valuable advise oncryosectioning, Cindy Wang for assistance with thesequencing, Gary Bernard for sharing unpublishedmicrospectrophotometric data on V. atalanta and two refereesfor constructive comments. Financial support came from theSwedish Research Council, Stockholm and the NationalScience Foundation (IBN-0346765).

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